Chemiluminescent 1,2-dioxetanes

ABSTRACT

Novel 1,2-dioxetanes with improved chemiluminescent properties, such as signal intensity, S/N ratio, T1/2, etc. are provided by spiroadamantyl 1,2-dioxetanes, wherein the remaining carbon atom of the ring bears an alkoxy, aryloxy, or arylalkoxy substituent, and either a phenyl or naphthyl ring, this aromatic ring bearing, at the meta position on the phenyl group, or a non-conjugated position on the naphthyl ring, a OX moiety wherein X is an enzyme-cleavable group, which when removed from the dioxetane, leaves the oxyanion which decomposies with chemiluminescence, the aryl ring further bearing an electron active substituent Z. The nature and placement of the Z substituent, at a position not adjacent the point of attachment to the dioxetane ring, strongly influences the properties of the dioxetane. Assays, as well as kits for the performance of those assays, include the dioxetane, an enzyme capable of cleaving the X group, and in certain cases, membranes and chemiluminscent enhancement agents.

This application is a CIP of U.S. application Ser. No. 08/231,673, filedApr. 25, 1994 now U.S. Pat. No. 5,582,980, which is a CIP U.S.application Ser. No. 08/057,903, filed May 7, 1993 now U.S. Pat. No.5,538,847.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to chemiluminescent 1,2-dioxetane derivativeswhich can be enzymatically activated to decompose and, throughdecomposition, release light. The dioxetanes are particularlycharacterized by the presence of an aromatic (phenyl or naphthyl) ringbonded to the dioxetane, which ring bears a meta-substituted or disjointenzymatically cleavable group, which when cleaved, leaves thephenoxyanion or naphthyloxyanion of the dioxetane, and, at the four orthe five position in the case of the phenyl, for example, an electrondonating or electron withdrawing group. By selecting the identity of thesubstituent at the four or five position (the Z moiety) particularaspects of the chemiluminescent properties of the dioxetane, includinghalf life, quantum yield, S/N ratio, etc., can be altered.

2. Background of the Invention

1,2-dioxetane enzyme substrates have been well established as highlyefficient chemiluminescent reporter molecules for use in enzymeimmunoassays and nucleic acid probe assays of a wide variety of types.These assays provide a preferred alternative to conventional assays thatrely on radioisotopes, fluorophores, complicated color shifting,secondary reactions and the like. Dioxetanes developed for this purposeinclude those disclosed in U.S. Pat. No. 4,978,614 as well as U.S. Pat.No. 5,112,960. U.S. Pat. No. 4,978,614 discloses, among others,3-(2'-spiroadamantane)4methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane,which has received world-wide attention, and is commercially availableunder the trade name AMPPD. U.S. Pat. No. 5,112,960, disclosescompounds, wherein the adamantyl stabilizing ring is substituted, ateither bridgehead position, with a variety of substituents, includinghydroxy, halogen, and the like, which convert the otherwise static orpassive adamantyl stabilizing group into an active group involved in thekinetics of decomposition of the dioxetane ring. Compounds of this typehave similarly received international attention, giving a faster andstronger signal than AMPPD in many applications. CSPD is aspiroadamantyl phenylphosphate dioxetane bearing a chlorine substituenton the adamantyl group, and, like AMPPD, is available from Tropix, Inc.of Bedford, Mass.

Compounds of this type have been particularly developed for enhancedsensitivity in assays for the presence of analytes in concentrations aslow as 10⁻¹² M and lower. In certain applications, compounds of thistype are used in conjunction with enhancers to detect analytes inconcentration of 10⁻¹² M or lower. These enhancement agents, whichinclude natural and synthetic water-soluble macromolecules, aredisclosed in detail in U.S. Pat. No. 5,145,772. Preferred enhancementagents include water-soluble polymeric quaternary ammonium salts, suchas poly(vinylbenzyltrimethylammonium chloride) (TMQ),poly(vinylbenzyltributylammonium chloride) (TBQ) andpoly(vinylbenzyldimethylbenzylammonium chloride) (BDMQ).

These enhancement agents improve the chemiluminescent signal of thedioxetane reporter molecules, apparently by providing a hydrophobicenvironment in which the dioxetane is sequestered. Water, an unavoidableaspect of most assays, due to the use of body fluids, is a natural"quencher" of the dioxetane chemiluminescence. The enhancement moleculesapparently exclude water from the microenvironment in which thedioxetane molecules, or at least the excited state emitter speciesreside, resulting in enhanced chemiluminescence. Other effectsassociated with the enhancer-dioxetane interaction could also contributeto the chemiluminescence enhancement.

Additional advantages can be secured by the use of selected membranes,including nylon membranes and treated nitrocellulose, providing asimilarly hydrophobic surface for membrane-based assays, and othermembranes coated with the enhancer-type polymers described.

Nonetheless, it remains a general goal of the industry to improve theperformance of these stabilized, chemiluminescent dioxetane reportermolecules, to improve the machine readability, sensitivity, andperformance aspects of the immunoassays, dependent on thechemiluminescent signal released by the dioxetanes.

By way of background, and as disclosed in all the patents referencedabove, the enzymatically-activated dioxetanes are used as reportermolecules, as substrates for enzymes which cleave the enzyme-labilegroup bonded to an aromatic substituent on the dioxetane ring. Thus, theenzyme, e.g., alkaline phosphatase is present alone or is covalentlylinked or otherwise complexed with either an antigen or antibody, inconventional antigen/antibody ligand binding assays, or a nucleic acidprobe in nucleic acid assays. The enzyme-bearing antigen or antibody, ornucleic acid probe, is then admixed with the analyte suspected ofcontaining the target antigen, or nucleic acid sequence, underconditions which permit complexing or hybridization between theantigen/antibody or probe/nucleic acid sequence. After washing away orseparating off all noncomplexed or nonhybridized material, the dioxetanesubstrate is added. If the suspected analyte is present, the enzyme willcleave the enzyme-labile group on the aromatic substituent on thedioxetane, e.g., phenyl or naphthyl, yielding the phenoxy or naphthyloxyanion intermediate. This anion decomposes, by electron transfer throughthe aromatic ring, cleaving the dioxetane ring, and yielding twocarbonyl-based products. The cleavage/decomposition event is thelight-releasing event.

To automate clinical assays, and to provide for substantial throughput,continued reductions in the half life, or T_(1/2) of the dioxetane, aswell as a reduction in the amount of time required to reach the maximumemission of light of the reporter molecule, is desirable. At the sametime, to detect analytes in extremely low concentrations, below, e.g.,about 10⁻¹² M, it is desirable to improve the intensity of the signal ofthe dioxetane reporter molecule, and simultaneously desirable to avoidincreasing the background noise due to nonenzymatically-induced lightrelease, so as to improve the overall sensitivity of the assay. Thus,further improvements in chemiluminescent dioxetane reporter moleculesare sought.

SUMMARY OF THE INVENTION

The above goals, and others, are met by a new class of dioxetanes,particularly characterized by a substituent on the aromatic ring bondedto the dioxetane, in addition to the meta-substituted enzyme-labilegroup. Thus, the novel dioxetanes of this invention have the generalizedstructure I, II or III below. ##STR1## wherein R is C1-12 alkyl,aralkyl, or aryl, preferably C1-4 alkyl, X is an enzyme labile groupcleavable by a specific enzyme which recognizes that group to leave thephenoxy or naphthoxy anion, and is preferably a phosphate, galactoside,or glucuronide. Y¹ and Y² are independently hydrogen, or an electrondonating or withdrawing group, and are preferably hydrogen, methoxy,carboxy or halogen, and most preferably one of Y¹ and Y² is hydrogenwhile the other is chlorine, and Z is an electron-active group, mostpreferably chlorine, alkoxy, alkyl or amido. When Z is on a phenyl ring,Z is in the four or five position. When OX and Z are substituted on anaphthyl group, OX is substituted such that the substitution isdisjoint, that is the total number of ring atoms between the point ofattachment to the dioxetane ring and the point of substitution,including the point of attachment and substitution, is an odd number, asdisclosed in U.S. Pat. No. 4,952,707. Substituent Z may be substitutedon the naphthyl ring at any position other than those adjacent the oneposition, or the point of attachment to the dioxetane ring.

By selecting the particular identity and location of Z, as anelectron-withdrawing or an electron-donating group, specificcharacteristics of the chemiluminescent behavior of the dioxetane,including its t1/2-chemiluminescence half lives, time to maximumemission, maximum emission wavelength, and chemiluminescent signalintensity can be affected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 compare the performance of disodium 3(4-methoxyspiro1,2-dioxetane-3,2'-(5'-chloro)tricyclo 3.3.1.1³.7 !decan!-4-yl)phenylphosphate dioxetane (CSPD) with a compound of this inventiondisodium-2-chloro-5-(4-methoxyspiro1,2-dioxetane-3,2'(5'-chloro-)tricyclo}3.3.1.1³,7 !-decan!-4yl)-phenylphosphate where the phenyl moiety bears a chlorine substituent at the 4position (CDP-Star). FIG. 2 reflects the presence of a chemiluminescenceenhancer, polyvinylbenzyltributylammonium chloride.

FIG. 3 is a comparison between CSPD and CDP-Star on a nylon membraneassay for biotinylated pBR322-35mer.

FIGS. 4 and 5 are reproductions of Kodak XAR-5 film exposures of westernblotting assays conducted on nylon and PVDF membranes comparing CSPD andCDP-Star.

FIGS. 6, 7 and 8 are reproductions of x-ray film contrasting CSPD andCDP-Star incubations of 10 minutes, 70 minutes and 19 hours,respectively, of an assay conducted on nylon membranes for the yeastgene RPB1.

FIG. 9 is a reproduction of x-ray film exposures reflectingchemiluminescent detection of DNA sequence ladders conducted on nylonmembranes contrasting CSPD and CDP-Star.

FIGS. 10 and 11 are electrophotographic duplications of x-ray filmimages of DNA sequencing obtained by use of the dioxetanes of theclaimed invention. These are compared against the current commercialstandard, CSPD.

FIGS. 12-21 are electrophotographic duplications of dot blot assayresults on membranes as indicated, employing dioxetanes of the claimedinvention, dioxetanes outside the scope of the claimed invention and thecommercial standards of CSPD and AMPPD. The membrane on which theseassays were conducted is set forth in the Figures.

DETAILED DESCRIPTION OF THE INVENTION

The dioxetanes of this invention are critically characterized by thesubstituents on the aromatic ring attached to the dioxetanes, which ringdetermines the electron transfer in the aryloxy anion, leading todecomposition and chemiluminescence. Thus, phenyl dioxetanes of theinvention have the following and generalized structure (I). ##STR2##

Thus, the adamantyl-stabilized dioxetanes of the claimed invention beartwo substituents on the phenyl ring in addition to the point ofattachment of the dioxetane, as well as 0, 1 or 2 non-hydrogensubstituents on the adamantyl ring. These substituents criticallycharacterize the electronic characteristics of the dioxetane, theoxyanion, and its decomposition behavior. The identities of eachsubstituent are set forth below.

R may be alkyl, aralkyl, cycloalkyl, or aryl, having 1-20 carbon atoms.R is preferably C1-C4 alkyl, more preferably C1-3 alkyl, most preferablymethyl. The identity of R may be optimized with regard to solubilityconcerns, where unusual analytes, or buffers, may pose particularproblems. Thus, R can be substituted or unsubstituted. If substituted, Rcan be substituted with 1-3 halo atoms such as chloro, diflouro,triflouro, dichloroflouro, an OSiE₃ moiety wherein E is an alkyl or arylof 1-12 carbon atoms, an alkoxy group, hydroxy lower alkyl group,carboxy group, sulfonyl group, amine, etc. as is generally disclosed inU.S. Pat. No. 5,225,584. Among preferred R groups is methyl, ethyl,propyl, flouro methyl, hydroxy ethyl, diflouroethyl and triflouroethyl.Each of Y¹ and Y² represent, individually, and independently hydrogen, ahydroxyl group, a halo substituent, a hydroxy lower alkyl group, a halolower alkyl group, a phenyl group, a halophenyl group, an alkoxy phenylgroup, an alkoxy phenoxy group, a hydroxyalkoxy group, a cyano group, anamide group, a carboxyl group or substituted carboxyl group, an alkoxygroup and other similar electron-active species. Preferred identitiesfor one of Y¹ and Y² are chlorine, hydroxy, and methoxy where the otheris hydrogen.

X is an enzyme-cleavable moiety. Thus, upon proper contact with asuitable enzyme, X is cleaved from the molecule, leaving the oxygenattached to the phenyl ring, and thus, the phenoxy anion. X is ideallyphosphate, galactoside, acetate, 1-phospho-2,3-diacylglyceride,1-thio-D-glucoside, adenosine triphosphate, adenosine diphosphate,adenosine monophosphate, adenosine, α-D-glucoside, β-D-glucoside,β-D-glucuronide, α-D-mannoside, β-D-mannoside, β-D-fructofuranoside,β-glucosiduronate, P-toluenesulfonyl-L-arginine ester, andP-toluenesulfonyl-L-arginine amide. X is preferably phosphate,galactoside or glucuronide, most preferably phosphate. It is importantto note that when substituted on the phenyl ring, OX is meta withrespect to the point of attachment to the dioxetane ring, that is, itoccupies the three position.

Z may occupy either the four or five position. Z is an electron-activesubstituent, the character of the electron-active species(electron-donating or electron-withdrawing), optimizing various aspectsof the dioxetane moiety. As an example, an electron-donating group, suchas a methoxy group, may enhance the dioxetane phenoxy aniondecomposition process, by facilitating the transferability of the freeelectrons from the aromatic ring O⁻ donor group, to the dioxetane ring.In contrast, an electron-withdrawing group would reduce or impair theability to transfer the free electrons to the dioxetane, thus slowingthe decomposition reaction and light emission, although ultimatelygiving a light signal of greater intensity. This should be contrastedwith the impact of the electron-withdrawing substituent on the adamantylgroup, such as chlorine, which substantially accelerates light emission,sharply reducing T_(1/2). Of surprising significance is the fact thatsubstitution in the six position is particularly undesirable. Suchsix-substituted phenyl dioxetanes exhibit extraordinarily fastdecomposition kinetics, and nearly no light emission. While Applicantsdo not wish to be restricted to this theory, it is believed that thisbehavior is due to steric considerations, that is, the ortho substituent"turns" the phenyl ring such that it destabilizes the dioxetane ring(destabilization through steric forces, not electron transfer) and asubstituent at the six position, e.g., methoxy, does not participate inelectron transfer. As discussed below, experiments involving6-substituted phenyl dioxetanes give essentially no signal.

The phenyl substituent on the dioxetane ring may instead be naphthyl(structures II and III) as ##STR3##

In the naphrhyl dioxetane, identities for R, Y¹ and Y², X and Z remainthe same. Instead of being restricted to the "meta" position, OX mayoccupy corresponding positions in the naphthyl ring, that is,non-conjugated positions, or positions such that the number of carbonatoms between the point of substitution and the point of attachment tothe dioxetane ring, including the carbons at both point of attachmentand point of substitution, are odd, as set forth in U.S. Pat. No.4,952,707. Phenyl meta-substituted dioxetanes, and naphthyl dioxetanessubstituted according to the pattern described above, may generally beexpected to give higher quantum yields than the corresponding para andconjugated systems.

As noted above, Z can be any electron-active substituent that does notinterfere with the chemiluminescent behavior of the dioxetane, and thuscan be selected from a wide variety of identities. Preferredelectron-active substituents include chloro, alkoxy (--OR), aryloxy(--OAr), trialkylammonium (--NR₃ +), alkylamido (--NHCOR, --NRCOR'),arylamido (--NHCOAr, --NRCOAr, --NArCOAr), arylcarbamoyl (--NHCOOAr,--NRCOOAr), alkylcarbamoyl (--NHCOOR, --NRCOOR'), cyano (--CN), nitro(--NO₂), ester (--COOR, --COOAr), alkyl- or arylsulfonamido (--NHSO₂ R,--NHSO₂ Ar), trifluoromethyl (--CF₃), aryl (--Ar), alkyl (--R),trialkyl-, triaryl-, or alkylarylsilyl (--SiR₃, SiAr₃, --SiArR₂), alkyl-or arylamidosulfonyl (--SO₂ NHCOR, --SO₂ NHCOAr), alkyl or aryl sulfonyl(--SO₂ R, SO₂ Ar) alkyl- or arylthioethers (--SR, SAr). The size of theZ substituent is generally limited only by solubility concerns. Wherereference is made to alkyl or R, R', etc., the alkyl moiety should have1-12 carbon atoms. Suitable aryl moieties include phenyl and naphthyl asexemplary moieties. Particularly preferred species include chloro andalkoxy.

Dioxetanes of the type described above, without the inclusion of the Zsubstituent, as previously noted, are disclosed in patents commonlyassigned herewith. Patents addressing dioxetanes of this type withoutthe inclusion of the Y and Z substituents have also been assigned toWayne State University, such as U.S. Pat. No. 4,962,192. Substitution ofthe Z substituent on the dioxetanes required development of thesynthesis of trisubstituted phenyl phosphonates which is describedbelow, under the title Novel Tri-substituted Phenyl 1,2-DioxetanePhosphates. The same general synthesis route can be employed fornaphthyl dioxetanes embraced herein, bearing in mind the substitutionpatterns required, as discussed above. The synthesis of these compoundsthrough the route described below involves the preparation of noveltri-substituted benzenes. Thus, as described below, an exemplarycompound involved in the synthesis of the dioxetanes of this classincludes 3-chloro-5-methoxybenzaldehyde. These tri-substituted compoundsconstitute key intermediates in a variety of synthetic pathways, the1,3,5 substitution pattern being a generally preferred and widelyapplicable pattern. It is Applicants' belief that these intermediateshave never previously been prepared, and are marked, in the synthesisroute described below, with an asterisk.

NOVEL TRI-SUBSTITUTED PHENYL 1,2-DIOXETANE PHOSPHATES

Synthesis

General. Commercial reagents were used as obtained without furtherpurification. Baker silica gels (60-200 mesh for gram scale, and 230-400mesh for milligram scale) were used for flash chromatography. ³¹ P NMRspectra were reported in parts per million relative to a phosphonc acidstandard. High resolution mass spectral analyses were run by J. L.Kachinski at Johns Hopkins University. Syntheses of dioxetanes 3 and 4were carried out following the procedure described below for dioxetanes1 and 2 respectively. Yields, melting points (uncorrected) and spectraldata are summarized for isolated intermediates.

3-Chloro-5-methoxy-4-trifluoromethanesulfonyloxy benzaldehyde (5)

A solution of 5-Cl-vanillin¹ (13.0 g, 70 mmol), chloroform (4 ml) andpyrridine (16 ml) was stirred at 0° C. Addition oftrifluoromethanesulfonic anhydride (12.4 ml, 75 mmol) at 0° C. over 30min gave clean formation of the triflate. The reaction mixture waspartitioned between EtOAc and 3N HCl. washed with dilute brine, driedover Na₂ SO₄, and evaporated under reduced pressure. Purification of theresulting yellow oil by silica gel chromatography (30% EtOAc/hexanes)yielded 18.5 g (83%) triflate 5 as yellow crystals.

IR (CHCl₃, cm⁻¹): 1705, 1590, 1461, 1425, 1225, 1205, 1132, 1049, 875,624

¹ H NMR (ppm): 3.99 (3H, s), 7.44 (1H, d, J=1.6 Hz), 7.57 (1H, d, J=1.7Hz), 9.92 (1H, s)

3-Chloro-5-methoxybenzaldehyde (6)

Triflate 5 (9 g, 28 mmol), palladium(II) acetate (120 mg, 0.5 mmol),1,1'-bis(diphenylphosphino)ferrocene (620 mg, 1 mmol) and hplc grade CH₃CN (10 ml) were mixed well in a teflon-lined stainless steel bomb. Afteradding freshly made, pulverized proton sponge formate² (7.84 g, 30mmol), the bomb was sealed and heated at 90° C. for 4 h. The cooledreaction was then filtered to remove proton sponge crystals, partitionedbetween EtOAc and 3N HCl. washed once each with dilute brine and diluteNaHCO₃, dried over Na₂ SO₄, and evaporated. Silica gel chromatography(15% EtOAc/hexanes) yielded 4.25 g (88.5%) of chloromethoxybenzaldehyde6, mp 45° C.

IR (CHCl₃, cm⁻¹): 2835, 1700 (C═O), 1590, 1576, 1461, 1425, 1380, 1320,1280, 1265, 1144, 1050, 850, 695

¹ H NMR (ppm): 3.84 (3H, s), 7.13 (1H, m), 7.26 (1H, m), 7.41 (1H, m),9.89 (1H, s)

Mass spectrum (EI, 70 eV): exact mass calcd for C₈ H₇ ClO₂ 170.0135,found 170.0134.

3-Chloro-5-methoxybenzaldehyde dimethyl acetal (7)

A methanol solution (20 ml) of benzaldehyde 6 (8.76 g, 51 mmol) wascleanly converted to dimethyl acetal 7 in the presence of trimethylorthoformate (5.62 ml, 51 mmol) and a catalytic amount ofp-toluenesulfonic acid. The reaction was quenched with triethylamine topH 7, evaporated to a small volume and partitioned between EtOAc andNaHCO₃. The organic layer was dried, evaporated under reduced pressureana purified by silica gel chromatography (10% EtOAc/hexanes) to give10.68 g (96%) of acetal 7 as a light yellow oil.

IR (CHCl₃, cm⁻¹): 2960, 2938, 2830, 1596, 1578, 1458, 1270, 1104,1050,989, 872, 865, 840

¹ H NMR (ppm): 3.31 (6H, s), 3.79 (3H, s), 5.31 (1H, s), 6.85 (1H, s),6.88 (1H, s), 7.04 (1H, s)

Diethyl 1-methoxy-1-(3-chloro-5-methoxyphenyl)methane phosphonate 8

Triethyl phosphite (3.2 ml, 19 mmol) was added dropwise to a solution ofacetal 7 (4.0 g, 18.5 mmol), boron trifluoride etherate (2.3 ml, 19mmol) and CH₂ Cl₂ (20 ml) at 0° C. After slowly warming the reaction toroom temperature (30 min), the solution was partitioned with diluteNaHCO₃, dried over Na₂ SO₄, evaporated and purified on silica gel(40%-100% EtOAc/hexanes) to give 4.6 g (77.5%) of phosphonate 8 as alight yellow oil.

IR (CHCl₃, cm⁻¹): 2990, 1591, 1573, 1458, 1254 (P═O), 1050 (P--O), 1025(P--O), 969, 870, 687

¹ H NMR (ppm): 1.24 (3H, t, J=7 Hz), 1.26 (3H, t, J=7 Hz), 3.37 (3H, s),3.78 (3H, s), 4.01-4.09 (4H, m), 4.40 (1H, d, J=16 Hz), 6.83 (1H, t, J=2Hz), 6.88 (1H, qt, J=2 Hz), 6.98 (1H, qt, J=2 Hz)

3-Chloro-5-methoxy-1-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (9)

Phosphonate 8 (4.62 g, 14 mmol) and 2-adamantanone (2.58 g, 17 mmol)were dissolved in anhydrous THF (35 ml) under argon and cooled to -68°C. Dropwise addition of lithium diisopropylamide (18.6 mmol) inanhydrous THF (20 ml) at -68° C. generated the ylid, followed bysubsequent olefination of the ketone. The reaction was slowly warmed toroom temperature over 2 h and then stirred at 75° C. for 1 h. Thesolution was partitioned between EtOAc/NH₄ Cl, dried over Na₂ SO₄,evaporated and purified by silica gel chromatography (2% EtOAc/hexanes),yielding 2.5 g (55%) of enol ether 9 as an oil.

¹ H NMR (ppm): 1.55-1.95 (12H, m), 2.61 (1H, br s), 3.21 (1H, br s),3.28 (3H, s), 3.78 (3H, s), 6.74 (1H, s), 6.80 (1H, s), 6.87 (1H, s)

3-Choloro-5-hydroxy-1-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidene-methyl)benzene (10)

Demethylation to enol ether phenol 10 proceeded cleanly upon heatingenol ether 9 (2.5 g, 7.8 mmol) in DMF (14 ml) at 155° C. in the presenceof sodium ethane thiolate (11.7 mmol). Upon cooling, the mixture waspartitioned between EtOAc and NH₄ Cl, dried over Na₂ SO₄ and evaporatedunder high vacuum to remove residual DMF. Chromatographic purification(silica gel, 20% EtOAc/hexanes) produced 2.3 g of phenol 10 as an oilwhich crystallized upon standing. Trituration of the solid with 5%EtOAc/hexanes gave white crystals, mp 133° C.

IR (CHCl₃, cm⁻¹): 3584 (OH), 3300 (OH), 2910, 1590, 1310, 1285, 1163,1096, 1080, 1011, 900, 840

¹ H NMR (ppm): 1.73-1.96 (12H, m), 2.62 (1H, brs), 3.20 (1H, brs), 3.32(3H, s), 5.65 (1H, brs), 6.73 (1H, m), 6.79 (1H, s)

Pyridinium 3-chloro-5-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)-1-phenyl phosphate (11)

Triethylamine (450 μl, 3.2 mmol) was added under an argon atmosphere toenol ether 10 (709 mg, 2.3 mmol) dissolved in anhydrous THF (10 ml). Thesolution was cooled to 0° C., at which time2-chloro-2-oxo-1,3,2-dioxaphospholane (Fluka, 285 μl, 3.0 mmol) wasadded dropwise. The reaction was warmed to room temperature, quicklypassed through an argon-flushed column under inert atmosphere to removetriethylammonium hydrochloride crystals. After rinsing the crystal cakeonce with THF, the solution was evaporated and pumped dry to give crudephospholane 11a.

Opening the phospholane ring upon reaction of 11a with NaCN (vacummdried, 179 mg, 3.65 mmol) in anhydrous DMF (6 ml) under argon, producedthe desired β-cyanoethyl diester phosphate 11b, as well as regeneratingenol ether phenol 10. Removal of DMF under high vacuum while warming theflask to 55° C., left a mixture of compounds 10 and 11b as ayellow-orange oil.

The above mixture was dissolved in methanol (8 ml) and stirred at 40° C.in the presence of NaOMe (1 ml of 4.25M NaOMe/MeOH, 6.4 mmol), effectingβ-elimination of the cyanoethyl group to give enol ether phosphate 11 asthe disodium salt. After evaporating the methanol, the solid wasdissolved in water and partitioned with minimal EtOAc to recover phenol10 (333 mg). Purification of the aqueous phase by preparative HPLC,using a CH₃ CN/H₂ O gradient through a polystyrene column (PLRP-S,Polymer Laboratories), followed by ion exchange with pyridiniumtoluenesulfonate (Amberlyst-IR 120+ resin) and lyophilization, yielded448 mg (78% over 3 steps accounting for recovered phenol) of enol etherphosphate 11 as a fluffy, off-white powder.

IR (CHCl₃, cm⁻¹): 2910, 1590, 1567, 1278, 1160, 1095, 945

¹ H NMR (ppm): 1.73-1.96 (12H, m), 2.63 (1H, brs), 3.20 (1H, brs), 3.32(3H, s), 5.89 (1H, s), 6.72 (1H, m), 6.79 (1H, t, J=2 Hz), 6.85 (1H, d,J=2 Hz)

³¹ P NMR (ppm): 54 (1P)

Disodium 3-chloro-5-(methoxyspiro 1,2-dioxetane-3,2'-tricyclo 3.3.1.1³.7!decan!-4-yl)-1-phenyl phospate (1)

A solution of enol ether phosphate 11 and5,10,15,20-tetraphenyl-21H,23H-porphine (TPP, 0.5 ml of a 2% solution inCHCl₃ by weight) in CHCl₃ (8 ml) was irradiated with a 250W highpressure sodium lamp at 10° C. while passing a stream of oxygen throughthe solution. A 5-mil piece of Kapton polyimide film (DuPont) placedbetween the lamp and the reaction mixture filtered out unwanted UVradiation. Analyticla HPLC (UV detect at 270 nm) showed completedioxetane formation upon irradiating 5 min. After evaporation of thechloroform at 0° C., the residue was dissolved in ice water in thepresence of Na₂ CO₃ (27 mg, 0.25 mmol) and purified by preparative HPLCas described above. The fractions were frozen and lyophilized at 0° C.,yielding 65.3 mg (90%) of dioxetane 1 as fluffy white powder. TLC of thedioxetane exhibited blue chemiluminescence by thermal decomposition uponheating. Enzymatic cleavage of the phosphate also inducedchemiluminescent decomposition in aqueous solutions.

¹ H NMR (D₂ O, ppm): 0.93 (1H, d, J=13 Hz), 1.21 (1H, d, J=13 Hz),1.44-1.69 (10H, m), 2.16 (1H, br s), 2.78 (1H, br s), 3.14 (3H, s), 7.20(2H, br s), 7.30 (1H, s)

³¹ P NMR (D₂ O, ppm): 24 (1P)

3-Chloro-5-hydroxy benzaldehyde dimethyl acetal (12)

5-Chloro-3-methoxy benzaldehyde dimethyl acetal (7, 3.21 g, 14.8 mmol)was demethylated with sodium ethane thiolate (19 mmol) in DMF (14 ml)while heating at 150° C. The resultant phenol 12 was cooled, partitionedbetween EtOAc and NH₄ Cl, dried over Na₂ SO₄, evaporated and pumped todryness on high vacuum to remove residual DMF. Chromatographicpurification (silica gel, 20% EtOAc/hexanes) afforded 2.75 g (92%) ofphenol 12 as a yellow oil. An analytical sample of the oil crystallizedupon further purification, mp 153° C.

IR (CHCl₃, cm⁻¹): 3580 (OH), 3325 (OH), 2490, 2830, 1599, 1585, 1449,1350, 1155, 1105, 1055, 894, 845

¹ H NMR (ppm): 3.32 (6H, s), 5.30 (1H, s), 5.73 (1H, br s), 6.81 (2H,m), 7.01 (1H, s)

3-chloro-5-pivaloyloxybenzaldehyde dimethyl acetal (13)

Phenol 12 (2.7 g, 13.3 mmol) and triethylamine (2.8 ml, 20 mmol) in CH₂Cl₂ (20 ml) were stirred at 0° C. Addition of trimethylacetyl chloride(1.64 ml, 13.3 mmol) cleanly yielded the pivaloyl ester. Standard workupprovided crude pivaloate 13 as an oil which was carried on the nextreaction without purification: no weight was taken. A small sample waspurified by prep TLC for spectral characterization.

IR (CHCl₃, cm⁻¹): 2980, 2940, 1749 (C═O), 1448, 1349, 1250, 1150, 1109,1056, 898

¹ H NMR (ppm): 1.34 (9H, s), 3.31 (6H, s), 5.36 (1H, s), 7.06 (2H, brs), 7.31 (1H, s)

Diethyl 1-methoxy-1-(3-chloro-5-pivaloyloxyphenyl)methane phosphonate(14)

A solution of acetal 13, boron trifluoride etherate (2.6 ml, 21 mmol)and CH₂ Cl₂ (10 ml) was stirred at -78° C. Addition of triethylphosphite (3.0 ml, 17.5 mmol) converted the acetal to phosphonate 14.Workup and purification (silica gel, 10% EtOAc/hexanes) yielded 2.43 goil (47% over 2 steps).

IR (CHCl₃, cm⁻¹): 2995, 2980, 1750 (C═O), 1600, 1581, 1442, 1247 (P═O),1110, 1028 (P--O), 975, 890

¹ H NMR (ppm): 1.22-1.26 (6H, d of t, J=2 Hz, 7 Hz), 1.31 (9H, s), 3.39(3H, s), 4.02-4.08 (4H, m), 4.44 (1H, d, J=16 Hz), 7.04 (2H, m), 7.27(1H, br s)

3-Chloro-5-pivaloyloxy-1-(methoxy-5-choloro-tricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (15)

Phosphonate 14 (2.4 g, 6.1 mmol) was dissolved in anhydrous THF (10 ml)under argon and cooled -68° C. Dropwise addition of lithiumdiisopropylamide (6.6 mmol) in anhydrous THF (7 ml) at low temperaturegenerated the ylid, evident by deep coloration. After 5 min, a THFsolution of 5-chloro-2-adamantanone (941 mg, 5 mmol) was added and thereaction was slowly warmed to room temperature over 40 min, followed byheating at 75° for 1 h to complete olefination. The solution waspartitioned between EtOAc/NH₄ Cl, dried over Na₂ SO₄ and evaporated togive a crude mixture of enol ether pivaloate 15 and the correspondingenol ether phenol 16. The crude oil was used without purification in thefollowing hydrolysis. A small sample was purified by prep TLC forspectral characterization.

IR (CHCl₃, cm⁻¹): 2935, 1750 (C═O), 1595, 1571, 1420, 1397, 1275, 1160,1110, 1024, 918, 906, 887, 829

¹ H NMR (ppm): 1.34 (9H, s), 1.68-1.78 (4H, m), 2.14-2.25 (7H, m), 2.77(1H, br s), 3.30 (3H, s), 3.42 (1H, br s), 6.88 (1H, d, J=1.5 Hz), 7.04(1H, m), 7.11 (1h, d, J=1.5 Hz)

3-Chloro-5-hydroxy-1-(methoxy-5-chloro-tricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (16)

Crude pivaloate 15 was hydrolyzed at room temperature with K₂ CO₃ (1.45g, 10.5 mmol) in 10 ml methanol. Evaporation of methanol, followed bystandard workup and purification (silica gel, 30% EtOAc/hexanes)afforded 1.095 g (63% over 2 steps) of a slightly yellow oil whichsolidified upon standing. Trituration of the solid produced whitecrystalline enol ether phenol 16, mp 130° C.

IR (CHCl₃, cm⁻¹): 3590 (OH), 3300 (OH), 2935, 1595, 1163, 1100, 1082,1030, 911

¹ H NMR (ppm): 1.69-1.83 (4H, m), 2.14-2.27 (7H, m), 2.77 (1H, br s),3.30 (3H, s), 3.41 (1H, br s), 5.21 (1H, br s), 6.67 (1H, d, J=1.5 Hz),6.81 (1H, m), 6.84 (1H, d)

Disodium 3-chloro-5-(methoxyspiro 1,2-dioxetane-3.2'-(5-chloro-)tricyclo3.3.1.1.sup.3.7 !-decan!-4-yl)-1-phenyl phospate (2)

Triethylamine (230 μl, 1.65 mmol) was added under an argon atmosphere toenol ether 16 (356 mg, 1.05 mmol) dissolved in anhydrous THF (5 ml). Thesolution was cooled to 0° C., at which time2-chloro-2-oxo-1,3,2-dioxaphospholane (Fluka, 143 μl, 1.55 mmol) wasadded dropwise. The reaction was warmed to room temperature and quicklypassed through an argon-flushed column under inert atmosphere to removetriethylammonium hydrochloride crystals. After rinsing the crystal cakeonce with THF, the solution was evaporated and pumped dry to give crudephospholane 17a.

Opening the phospholane ring upon reaction with NaCN (vacuum dried, 69mg, 1.4 mmol) in anhydrous DMF (5 ml) under argon, produced the desiredβ-cyanoethyl diester phosphate 17b. Removal at DMF under high vacuumwhile warming the flask to 55° C. left the crude diester phosphate as anorange oil.

A solution of cyanoethyl phosphate 17b and5,10,15,20-tetraphenyl-21H,23H-porphine (TPP, 1.5 ml of a 2% solution inCHCl₃ by weight) in CHCl₃ (10 ml) was irradiated with a 250W, highpressure sodium lamp at 10° C. while passing a stream of oxygen throughthe solution. A 5-mil piece of Kapton polyimide film (DuPont) placedbetween the lamp and the reaction mixture filtered out unwanted UVradiation. Analytical HPLC (UV detector at 270 nm) showed completedioxetane formation upon irradiating 15 min. After evaporation of thechloroform at 0° C. the residue was dissolved in methanol anddeprotected to the disodium phosphate dioxetane with NaOMe (0.5 ml of4.25M NaOMe/MeOH, 2 mmol). Upon β-elimination of the cyanoethyl group,the solvent was evaporated at 0° and the residue dissolved in ice water.Purification by preparative HPLC. as described above, followed bylyophilization at 0° C., yielded 289 mg (60% over 4 steps) of dioxetane2 as a fluffy white powder.

¹ H NMR (D₂ O, ppm, mixture of syn/anti isomers): 0.86 (1H, d), 1.13(1H, d, J=14 Hz), 1.30 (1H, d), 1.37 (1H, d), 1.45-2.07 (18H, m), 2.27(1h, br s), 2.32 (1H, br s), 2.95 (2H, br s), 3.09 (3H, s), 3.11 (3H,s), 7.0-7.3 (4H, br s), 7.25 (1H, s), 7.28 (1H, s)

3,5-Dimethoxybenzaldehyde dimethyl acetal (18)

IR (CHCl₃, cm⁻¹): 2958, 2935, 1598, 1460, 1426, 1357, 1190, 1154, 1101,1053, 840

¹ H NMR (ppm): 3.32 (6H, s), 3.78 (6H, s), 5.28 (1H, s), 6.41 (1H, m),6.60 (2H, m)

3-Hydroxy-5-methoxybenzaldehyde dimethyl acetal (19)

IR (CHCl₃, cm⁻¹) 3590 (OH), 3345 (OH), 2940, 2830, 1600, 1462, 1432,1355, 1190, 1150, 1110, 1055, 841

¹ H NMR (ppm): 3.32 (6H, s), 3.77 (3H, s), 5.28 (1H, s), 6.37 (1H, d,J=2 Hz), 6.53 (1H, br s), 6.58 (1H, br s)

3-Methoxy-5-pivaloyloxybenzaldehyde dimethyl acetal (20)

(73% over 3 steps, oil)

IR (CHCl₃, cm⁻¹): 2960, 2935, 1741 (C═O), 1608, 1597, 1462, 1350, 1273,1190, 1139, 1115, 1056, 999, 902, 848

¹ H NMR (ppm): 1.34 (9H, s), 3.31 (6H, s), 3.80 (3H, s), 5.35 (1H, s),6.57 (1H, d, J=2 Hz), 6.75 (1H, br s), 6.87 (1H, br s)

Diethyl 1-methoxy-1-(3-methoxy-5-pivaloyloxyphenyl)methane phosphonate(21)

(40%, oil)

IR (CHCl₃, cm⁻¹): 2990, 2980, 1742 (C═O), 1606, 1590, 1463, 1272, 1240,1136, 1110, 1100, 1055, 1023, 970

¹ H NMR (ppm): 1.21 (3H, t, J=3 Hz), 1.23 (3H, t), 1.32 (9H, s), 3.39(3H, s), 3.78 (3H, s), 4.06 (4H, m), 4.44 (1H, d, J=16 Hz), 6.56 (1H,m), 6.72 (1H, m), 6.85 (1H, m)

3-Methoxy-5-pivaloyloxy-1-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (22a).

IR (CHCl₃, cm⁻¹): 2910, 1740 (C═O), 1600, 1580, 1560, 1325, 1272, 1140,1114, 1097, 1079, 1055

¹ H NMR (ppm): 1.35 (9H, s), 1.56-1.96 (12H, m), 2.68 (1H, br s), 3.23(1H, br s), 3.31 (3H, s), 3.80 (3H, s), 6.53 (1H, t, J=2 Hz), 6.61 (1H,br s), 6.72 (1H, m)

3-Hydroxy-5-methoxy-1-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (22).

(64%, white crystals mp 159° C).

IR (CHCl₃, cm⁻¹): 3590 (OH), 3320 (OH), 2910, 1591, 1342, 1150, 1098

¹ H NMR (ppm): 1.78-1.97 (12H, m), 2.68 (1H, br s), 3.23 (1H, br s),3.33 (3H, s), 3.78 (3H, s), 5.49 (1H, s), 6.37 (1H, m), 6.45 (2H, m)

Pyridinium 5-methoxy-3-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)-1-phenyl phosphate (23).

(62%, off-white fluffy powder)

IR (CHCl₃, cm⁻¹): 2911, 1584, 1448, 1425, 1328, 1149, 1099, 960, 870

¹ H NMR (ppm): 1.68-1.92 (12H, m), 2.63 (1H, br s), 3.17 (1H, br s),3.23 (3H, s), 3.68 (3H, s), 6.55 (1H, br s), 6.72 (1H, br s), 6.76 (1H,br s), 6.98 (1H, br s)

Disodium 5-methoxy-3-(methoxyspiro 1.2-dioxetane-3.2'-tricyclo3.3.1.1³.7 !-decan!-4-yl)-1-phenyl phosphate (3).

(85%, white fluffy powder)

¹ H NMR (D₂ O, ppm): 0.98 (1H, br d), 1.22 (1H, br d), 1.46-1.76 (10H,m), 2.20 (1H, br s), 2.78 (1H br s), 3.14 (3H, s), 3.74 (3H, s), 6.91(1H, br s), 6.68-6.97 (2H, very broad signal)

³¹ P NMR (D₂ O, ppm): 44.8 (1P)

3-Hydroxy-5-methoxy-1-(methoxy-5-chloro-tricyclo 3.3.1.1³.7!-dec-2-ylidenemethyl)benzene (24).

(63%, white crystals, mp 134° C.)

IR (CHCl₃, cm⁻¹): 3590 (OH) 3330 (OH), 2930, 1610, 1591, 1450, 1430,1341, 1150, 1100, 1080, 1056, 1028, 829

¹ H NMR (ppm): 1.68-2.40 (11H, m), 2.82 (1H, br s), 3.31 (3H, s), 3.42(1H, br s), 3.78 (3H, s), 6.37--6.41 (3H, m)

Disodium 5-methoxy-3-(methoxyspiro1,2-dioxetane-3.2'-(5-chloro-)tricyclo 3.3.1.1.sup.3.7!decan!-4-yl)-1-phenyl phosphate (4).

(57% over 4 steps, white fluffy powder)

¹ H NMR (D₂ O, ppm, mixture of syn/anti isomers): 0.94 (1H, br d), 1.19(1H, br d), 1.42 (1H, br d), 1.50 (1H, br s), 1.58 (1H, br d), 1.67-2.16(17H, m), 2.38 (1H, br s), 2.40 (1H, br s), 3.00 (2H, br s), 3.15 (3H,s), 3.16 (3H, s), 3.73 (3H, s), 3.74 (3H, s), 6.90 (1H, br s), 6.93 (1H,br s), 6.65-7.00 (4H, very broad signal)

³¹ P NMR (D₂ O, ppm, mixture of syn/anti isomers): 44.8 (2P)

References

1. 5-Chlorovanillin was synthesized as described by Hann and Spencer (J.Am. Chem. Soc., 1927, 49:535-537), mp 163° C.

2. Proton sponge formate(N,N,N',N',-tetramethyl-1,8-naphthalenediammonium formate): Formic acid(98%, 1.2 ml, 31 mmol) was added to a solution of proton sponge (6.8 g,32 mmol) and CH₂ Cl₂ (8 ml) at 0° C. After warming to room temperature,the solvent was evaporated and the proton sponge formate crystallized aswhite crystals while drying on high vacuum with minimal warming. Protonsponge formate crystals (mp 79° C.) must be used soon after preparationsince formic acid will evaporate upon standing, leaving proton sponge(mp 50° C).

3-Methoxy-5-nitro-4-hydroxy benzaldehyde dimethyl acetal (25)

A methanol solution (30 ml) of 5-nitrovanillin (5.0 g, 97%, 18.4 mmol)was cleanly converted to dimethyl acetal 25 in the presence of trimethylorthoformate (2.8 ml, 25 mmol) and a catalytic amount ofp-toluenesulfonic acid. The reaction was quenched with triethylamine topH 8, evaporated to a small volume and partitioned between EtOAc andNaHCO₃. The aqueous layer was washed once with EtOAc. The organic layerswere dried over Na₂ SO₄, decanted and evaporated to an orange-red oilthat crystallized upon pumping. Recrystallization from 50% EtOAc/hexanesgave 5.55 g (93) acetal 25 as red-orange crystals, mp 58°-59° C.

IR (CHCl₃, cm⁻¹): 3300, 3010, 2930, 2820, 1620, 1543, 1460, 1445, 1392,1341, 1320, 1254, 1132, 1101, 1058, 990, 865

¹ H NMR (ppm): 3.31 (6H, s), 3.94 (3H, s), 5.31 (1H, s), 7.22 (1H, d,J=1.7 Hz), 7.78 (1H, d)

3-Methoxy-5-nitro-4-trifluoromethanesulfonyloxy benzaldehyde dimethylacetal (26)

A solution of dimethyl acetal 25 (5.0 g, 20.6 mmol), chloroform (3 ml)and pyridine (8 ml) was stirred at 0° C. under argon. Addition oftrifluoromethanesulfonic anhydride (4.0 ml, 23.8 mmol) at 0° C. over 10min, followed by stirring at room temperature overnight gave cleanformation of the triflate. The solvents were evaporated under highvacuum while warming the oil to 45° C. and traces of pyridine werechased with 4 ml toluene. The resulting oil was pumped well under highvacuum, taken up in 50% EtOAc/hexanes and triturated with 50%EtOAc/hexanes to separate the desired triflate (in solution) from thefine pyridinium triflate crystals. Evaporation of the triturationsolution, followed by purification of the oil on a silica gel column,eluting with 30% EtOAc/hexanes, yielded 6.43 g (84) of triflate 26 as alight yellow oil.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm): 3.35 (6H, s), 4.00 (3H, s), 5.42 (1H, s), 7.42 (1H, d,J=1.6 Hz), 7.73 (1H, d)

3-Methoxy-5-nitro-benzaldehyde dimethyl acetal (27)

5-Nitrophenyl triflate 26 (7 g, 18.7 mmol), palladium (II) acetate (88mg, 0.39 mmol), 1,1'-bis(diphenylphosphino)ferrocene (430 mg, 0.78 mmol)and hpic grade CH₃ CN (10 ml) were mixed well in a teflon-linedstainless steel bomb. After adding freshly made, pulverized protonsponge formate (5.1 g, 19.6 mmol), the bomb was sealed and heated at 90°C. for 2 h. The reaction mixture was take up in EtOAc, passed through asilica gel plug, and then purified on a silica gel column, eluting with0-30% EtOAc/hexanes to yield 1.5 g (35%) methoxynitrobenzaldehyde acetal27.

IR (CHCl₃, cm⁻¹): 3005, 2960, 2935, 2835, 1532 (--NO₂), 1463, 1450, 1343(--NO₂), 1280, 1190, 1158, 1055, 990, 871

¹ H NMR (ppm): 3.33 (6H, s), 3.89 (3H, s), 5.41 (1H, s), 7.33 (1H,s),7.68 (1H, s), 7.92 (1H, s)

Diethyl 1-methoxy-1-(3-methoxy-5-nitrophenyl)methane phosphonate (28)

Triethyl phosphite (0.98 ml, 5.7 mmol) was added dropwise to a solutionof dimethyl acetal 27 (1.08 g, 4.7 mmol), boron trifluoride etherate(1.2 ml, 9.8 mmol) and CH₂ Cl₂ (10 ml) at 0° C. After warming thereaction to room temperature overnight, the solution was partitionedwith 3N HCl and the aqueous layer was washed with CH₂ Cl₂ twice. Theorganic layers were washed with dilute NaHCO₃, dried over Na₂ SO₄,decanted and evaporated. The crude residue was purified on a silica gelcolumn, eluting with 0-80% EtOAc/hexanes, to give 1.36 g (86%)phosphonate 28 as a slightly yellow oil.

IR (CHCl₃, cm⁻¹): 2995, 1532 (--NO₂), 1350 (--NO₂); 1280, 1258, 1243,1096, 1053, 1025, 973, 721

¹ H NMR (ppm): 1.28 (6H, t, J=7.1 Hz), 3.44 (3H, s), 3.90 (3H, s),4.08-4.15 (4H, m), 4.55 (1H, d, J=16 Hz), 7.34 (1H, d), 7.69 (1H, d,J=2.1 Hz), 7.87 (1H, d, J=1.6 Hz)

Diethyl 1-methoxy-1-(3-amino-5-methoxyphenyl)methane phosphonate (29)

Nitro phosphonate 28 is dissolved in methylene chloride and added to a1M NaOH solution containing nBu₄ NBr and sodium hydrosulfite. Thebiphasic solution is stirred vigorously, with warning if necessary,until reduction of the nitro substituent to aniline 29 is complete. Thecooled solution is partitioned between CH₂ Cl₂ and minimal water, andthe aqueous layer is washed with CH₂ Cl₂ as needed to obtain the crudeaniline. The combined organic layers are dried, decanted and evaporated.The residue is then passed through a short silica gel plug to giveaniline 29.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm):

(References for other reduction conditions are appended to the synthesissummary.)

Diethyl 1-methoxy-1-(3-methoxy-5-trifluoroacetamidophenyl)methanephosphonate (30)

Phosphonate 29 is quantitatively acetylated by addition oftrifluoroacetic anhydride (1 eq) and triethylamine (1.3 eq) in 10 ml CH₂Cl₂ at 0° C. Evaporation of solvents, followed by silical gel columnpurification yields trifluoroacetamide 30.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm):

3-Methoxy-5-trifluoroacetamido-1-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (31)

Phosphonate 30, dissolved in anhydrous THF, is cooled to -68° C. underan argon atmosphere. Similarly, 2-adamantanone (1.1 eq) is dissolved inanhydrous THF and cooled to -68° C. under argon in a separate flask. Tothe phosphonate solution is added 2.5M nBuLi is added to complete thered color of the ylid persists. At this point, 1.2 eq nBuLi is added tocomplete the ylid formation and the resulting colored solution isstirred at -68° C. for 5 min. While maintaining the low temperature,2-adamantanone in THF is slowly added to the ylid over an hour. Afterthe final addition of ketone, the reaction mixture is stirred for 2 hwhile warming to room temperature. The reaction is then heated at refluxfor 1 h, cooled and quenched by partitioning with EtOAc and saturatedNH₄ Cl. The organic layer is dried over Na₂ SO₄ and chromatographed withEtOAc/hexanes on a silica gel column to give enol ether 31.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm):

3-Amino-5-methoxy-1-(methoxytricyclo 3.3.1.³.7!dec-2-ylidenemethyl)benzene (32)

Trifluoroacetamide enol ether 31 is hydrolyzed at 60° C. with finelyground K₂ CO₂ (3 eq) in MeOH containing trace water. Work up bypartitioning the mixture with EtOAc/H₂ O, followed by silica gelchromatography provides enol ether aniline 32.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm):

3-Carbamoyl-5-methoxy Derivatives (3-NHCO₂ X)3-para-Methoxyphenylcarbamoyl-5-methoxy-1-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (33)

Enol ether aniline 32 in methylene chloride is carboxylated with4-methoxyphenyl chloroformate (1.1 eq) in the presence of triethylamine(2.0 eq) at 0° C. The reaction mixture is partitioned with CH₂ Cl₂ /H₂O, washed with dilute NaHCO₃, dried ever Na₂ SO₄, evaporated andchromatographed on silica gel to yield enol ether p-methoxyphenylcarbamate 33.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm):

3-tert-Butylcarbamoyl-5-methoxy-1-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (34)

A methylene chloride solution of enol ether aniline 32, triethylamine(1.5 eq) and BOC-ON (1.3 eq) is stirred at 55° C. in a tightly cappedKimax tube to effect t-butyl carbamate formation. The solution iscooled, evaporated to small volume and, upon addition of MeOH to theresidue, the desired carbamate 34 precipitates.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm):

3-N-Sulfonamido-5-methoxy Derivatives (3-NHSO₂ X)3-N-Toluenesulfonamido-5-methoxy-1-(methoxtricyclo 3.3.1.1³.7!dec-2-ylidenemethyl) (35)

A methylene chloride solution of enol ether aniline 32 is sulfonylatedwith tosyl chloride (1.1 eq) in the presence of triethylamine (2.0 eq)at 0° C. The reaction mixture is partitioned with CH₂ Cl₂ /H₂ O, washedwith dilute NaHCO₃, dried over Na₂ SO₄, evaporated and chromatographedon silica gel to yield N-toluenesulfonamido enol ether 35.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm):

3-N-Trifluoromethylsulfonamido-5-methoxy-1-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (36)

A methylene chloride solution of enol ether aniline 32 is sulfonylatedwith trifluoromethylsulfonic anhydride (1.11 eq) at 0° C. The reactionmixture is partitioned with CH₂ Cl₂ /H₂ O, dried over Na₂ SO₄,evaporated and chromatographed on silica gel to yieldN-trifluoromethylsulfonamido enol ether 36.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm):

3-Amido-5-methoxy Derivatives (3-NHCOX)3-N-Benzamido-5-methoxy-1-(methoxytricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)benzene (37)

A pyridine solution of enol ether aniline 32 is reacted with benzoylchloride (1.1 eq) at 0° C. The solvent is evaporated and pumped well toyield a crude oil, which is partitioned between CH₂ Cl₂ /H₂ O, dried andevaporated. Chromatography on silica gel yields benzamido enol ether 37.

IR (CHCl₃, cm⁻¹):

¹ H NMR (ppm):

The 3-nitrogen-substituted phenyl enol ethers (compounds 33-37) aredemethylated with sodium ethane thiolate, and then phosphorylated andphotooxygenated ad described for dioxetanes 1 and 2 to obtain theanalogous dioxetanes.

Among other inventive compounds within the structure of formula I, aparticularly preferred compound has the structure: ##STR4##

The name of this compound is disodium 2-chloro-5-(4-methoxyspiro1,2-dioxetane-3,2'-(5'-chloro-)tricyclo 3.3.1.1³.7!decan!-4-yl)-1-phenyl phosphate.

This compound is generally referred to as CDP-Star. It can besynthesized as shown in the following reaction scheme. ##STR5##

4-Chloro-3-methoxybenzaldehyde dimethyl acetal 3

A heterogenous mixture of methanol (2 ml), CH₂ Cl₂ (3 ml),4-chloro-3-methoxybenzaldehyde (2 g, 11.7 mmol; prepared essentially asdescribed by R. M. Riggs et al., J. Med. Chem., 30 1887, 1987.),trimethyl orthoformate (1.7 ml, 15.5 mmol) and a large crystal ofp-toluenesulfonic acid was stirred at room temperature for one hour.Additional MeOH (1 ml) and a crystal of p-toluenesulfonic acid wereadded and the solution was warmed until homogenous. Upon completion ofthe reaction, the solution was stirred 5 min with excess solid NaHCO₃and rotory evaporated to remove solvents. The paste was dissolved in 40ml EtOAC, partitioned against dilute NaHCO₃ solution, and evaporated toyield a light brown oil. The reaction was repeated with another 2 g of4-chloro-3-methoxybenzaldehyde and both product oils were combined togive 4.37 g (86%) of dimethyl acetal 3.

IR (neat, cm⁻¹): 2930, 2810, 1582, 1580, 1483, 1460, 1402, 1348, 1268,1100, 1059, 989, 861, 827, 790

¹ H NMR (CDCl3, ppm): 3.30 (6H, s), 3.90 (3H, s), 5.33 (1H, s), 6.95(1H, d), 7.03 (1H, s), 7.32 (1H, d)

Diethyl 1-methoxy-1-(4-chloro-3-methoxyphenyl)methane phosphonate 4

A solution of dimethyl acetal 3 (4.3 g, 20 mmol), sieve-dried CH₂ Cl₂(20 ml) and triethyl phosphite (4.1 ml, 24 mmol) was stirred under argonat -78° C. Boron trifluoride ethereate (2.95 ml, 24 mmol) was addeddropwise at -78° C., the solution was stirred 5 min and stored overnightat -20° C. The next day the reaction was warmed to room temperature andstirred 5 hours to complete phosphonate formation. With vigorousstirring, the reaction was quenched with solid NaHCO₃ followed by 40 mlsaturated NaHCO₃ solution. After gas evolution ceased, 40 ml CH₂ Cl₂ and20 ml H₂ O were added, the biphasic mixture was partitioned and the CH₂Cl₂ phase was recovered, dried over Na₂ SO₄ and evaporated. Afterpumping in vacuo, the oil was purified on a silica gel plug, elutingwith CH₂ Cl₂ to yield phosphonate 4 as a light yellow oil (6.01 g.,99%).

IR (neat, cm⁻¹): 2980, 2930, 1590, 1580, 1480, 1460, 1408, 1280, 1250,1095, 1055, 1025, 967, 871, 809, 790, 754, 728

4-Chloro-3-methoxy-1-(methoxy-5-chloro-tricyclo 3,3,1,1³.7!dec-2-ylidenemethyl)-benzene 5

Phosphonate 4 (3.2 g, 10 mmol) was dissolved in 30 ml dry THF underargon and cooled to -78° C. Dropwise addition of nBuLi (2.3M, 4.4 ml,10.1 mmol) generated a yellow-orange phosphonate ylid. After stirringthe ylid solution for 10 min, 5-chloro-2 adamantanone (1.75 g, 9.5mmol), dissolved in 8 ml THF, was added dropwise to the ylid at -78° C.The reaction was slowly warmed to room temperature over 45 min andrefluxed for 2 h. Upon cooling, the THF was stripped in vacuo and theproduct was partitioned between EtOAc/hexanes (1:1) and dilute NaHCO₃.The organic layer was dried over NaHCO₃. The organic layer was driedover Na₂ SO₄, stripped of solvent and purified on silica gel (2-4%EtOAc/hexanes) to give 3.3 g (96%) of enol ether 5 as a colorless,viscous gum.

4-Chloro-3-hydroxy-1-(methoxy-5-chloro-tricyclo 3.3.1.1³.7!dec-2-ylidenemethyl)-benzene 6

Demethylation to enol ether phenol 6 proceeded cleanly upon heating enolether 5 (3.3 g, 9.3 mmol) in 22 ml DMF at 135° C. in the presence ofsodium ethanethiolate (14 mmol) for 1.5 h. The reaction was cooled andpartitioned between 50 ml EtOAc, 100 ml 1M NH₄ Cl and 10 ml saturatedNaHCO₃ solution. The organic phase was recovered and washed well withwater while the aqueous phase was washed once with EtOAc. The EtOAclayers were combined, washed with brine, dried over Na₂ SO₄ and strippedof solvent in vacuo. The crude oil was purified on a silica gel column,eluting with 50% CH₂ Cl₂ /hexanes, to give 3.6 g of phenol 6 as a crudeoil. Further purification by two crystallizations from a cooled 15% CH₂Cl₂ /hexanes solution provided phenol 6 as a solid (2.18 g, 68%).

IR (CHCl₃, cm⁻¹): 3530 (OH), 3300 (OH), 2920, 2845, 1568, 1478, 1308,1190, 1166, 1090, 1079, 1042, 1020, 821

¹ H NMR (CDCl3, ppm): 1.57-2.28 (11H, m), 2.75 (1H, br s), 3.27 (3H, s),3.41 (1H, br s), 5.57 (1H, s), 6.79 (1H, dd, J=8 Hz, 2 Hz), 6.93 (1H, d,J=2 Hz), 7.28 (1H, d, J=8 Hz)

Sodium 2-cyanoethyl 2-chloro-5-(methoxy-(5-chloro)tricyclo 3,3,1,1³,7!dec-2-ylidenemthyl)-1-phenyl phosphate 7

To a solution of phenol 6 (0.75 g 2.2 mmol), triethylamine (400 μl, 2.86mmol) and anhydrous THF (8 ml) was added to2-chloro-2-oxo-1,3,2-dioxaphospholane (Fluka, 240 μl, 2.6 mmol) at roomtemperature under argon. The reaction was stirred for 3 hours, duringwhich time triethylammonium hydrochloride precipitated out. The reactionsolution was pipetted off the precipitate with a cotton-tipped syringeunder a strong flow of argon. The precipitate was rinsed several timeswith ether and the combined solution and rinses were evaporated in vacuoto give a foam, which was protected from exposure to moisture.

The foam was dissolved in anhydrous DMF (4 ml) and stirred with dry NaCN(140 mg, 2.8 mmol) at room temperature for 24 h to form the β-cyanoethylphosphate diester. The reaction mixture was pumped at high vacuum at 55°C. to remove DMF, leaving phosphate diester 7 as a gum. The crudephosphate diester was photooxygenated without further purification.

Syn- and Anti-disodium 2-chloro-5-(4-methoxyspiro1,2-dioxetane-3,2'-(5'-chloro-)tricyclo 3,3,1,1³,7!-decan!-4-yl)-1-phenyl phosphate 1

Phosphate diester 7 was dissolved in 20 ml 10% MeOH/CHCl₃ to which wasadded 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP, 0.8 ml of a 2 mg/mlCHCl₃ solution). The reaction mixture was saturated with oxygen andirradiated with a 250W, high pressure sodium vapor lamp wrapped withKapton film at 5° C. while passing oxygen through the solution.Analytical reverse phase HPLC analysis showed complete dioxetaneformation upon irradiating 20 min. The solvents were stripped in vacuoat room temperature, the residue was pumped to a gum under high vacuum,and stored at -20° C.

The crude cyanoethyl phosphate diester dioxetane 8, dissolved in 11 mlMeOH, was deprotected with sodium methoxide (0.5 ml of 25%, weight NaOMein MeOH) at room temperature for 30 min. Upon completion ofβ-elimination of the cyanoethyl group, 2 ml saturated NaHCO₃ solutionwas added and the MeOH was rotory evaporated. HPLC grade water (15 ml)was added and the brown solution was passed through a 0.45μ nylonfilter. The solution volume was adjusted to 40 ml with HPLC grade waterand purified by preparative HPLC, using a CH₃ CN/H₂ O gradient through apolystyrene column (PLRP-S, Polymer Laboratories). The freeze-driedfractions yielded 0.81 g (74% from phenol 6) of dioxetane 1. Analyticalreverse phase HPLC on a polystyrene column using a gradient ofacetonitrile and 0.1% NaHCO3 and UV detection at 270 nm, showed one peakwith a front running shoulder which represented a mixture syn and antiisomers. A sample of the product, as an isomer mixture, was dissolved ina 0.1M diethanolamine buffer (1 mM MgCl₂) at pH 10. Upon being treatedwith alkaline phosphatase, light was emitted as expected, thusconfirming the product as a 1,2-dioxetane of the entitled structure.

EXAMPLES

Various dioxetanes within the scope of this invention have been preparedand tested for essential properties. Included as prepared and testeddioxetanes are those where R is methyl, X is phosphate and Y² ischlorine, and the Z is at the 4 or 5 position on a phenyl ring. In thetests below, those dioxetanes are compared against commercial standardsCSPD® and AMPPD™.

Chemiluminescent Detection of Alkaline Phosphatase in Solution

Alkaline phosphatase (5.25×10⁻¹⁷ moles) was incubated in 0.5 ml of 0.1Mdiethanolamine, 1 mM MgCl₂, pH 10, containing 0.4 mM dioxetane at roomtemperature. The chemiluminescence (5 second integral) was measured in aBerthold LB952T luminometer at 5, 10, 20, 30, 44, 50 and 60 minutes.FIGS. 1 and 2 compare the performance of CSPD® and CDP-Star™. FIG. 1shows the comparison of CSPD® and CDP-Star™ plotted as relative lightunits (RLU) vs time. The average of three replicates are plotted. FIG. 2shows the results obtained from another set of samples containing 1mg/ml of the chemiluminescence enhancing polymerpolyvinylbenzyltributylammonium chloride.

Chemiluminescent Detection of Biotinylated pBR322-35mer on NylonMembrane

Biotinylated pBR322 35-mer (13.1 pg in 1 μl) was spotted onto a smallpiece of positively charged nylon membrane (Tropilon-Plus™). Themembrane was dried completely and DNA was fixed to the membrane by UVcross-linking (120 mJ/cm²). The membrane was wetted with PBS and thenincubated in Blocking Buffer (0.2% 1-Block™, 0.5% SDS in PBS) for 10minutes, in streptavidin-alkaline phosphatase conjugate (Avidx-AP™,Tropix; diluted 1:5000 in Blocking Buffer) for 20 minutes, and washedonce for 5 minutes with Blocking Buffer and three times for 5 minutewith Wash Buffer (0.5% SDS in PBS). Membranes were then washed twice for5 minutes with Assay Buffer (0.1M DEA, 1 mM MgCl₂, pH 10.0). Finally,membranes were trimmed and sealed in a small square of heat-sealableplastic with 40 μl of 0.25 mM CSPD or CDP-Star (diluted in AssayBuffer). The sealed piece of membrane was immediately taped to a tube(pre-incubated at 22° C.) and placed in a Turner Model 20e luminometer(Turner Designs, Inc. Mountain view, Calif.). Light emission wasrecorded every 5 minutes, for a period of 24 hrs at 22° C. FIG. 3 is aplot of the chemiluminescence intensity (RLU) vs time for CSPD andCDP-Star.

Chemiluminescent Detection of Western Blotted Human Transferrin

Purified human transferrin (Boehringer/Mannheim cat #1317 415) wasserially diluted with 1× SDS-PAGE loading buffer (0.06M Tris-HCl, pH6.8, 2.25% glycerol, 0.5% β-mercaptoethanol, 2% SDS, 0.05% bromophenolblue). Dilutions were heated at 95° C. for 5 minutes and 5 μl of dilutedsample was loaded per gel lane. Samples were electrophoreticallyseparated by SDS-PAGE on 10% polyacrylamide minigels, using a HoeferSE250 minigel apparatus. Each blot contains 10, 3.3, 1.1, 0.37, 0.12 and0.04 ng amounts of transferrin. Following electrophoresis, gels andmembranes were equilibrated with transfer buffer (5 mM MOPS pH 7.5, 2 mMsodium acetate, 20% MeOH) for 15 minutes. Protein was transferred toPVDF (Tropifluor™) and positively charged nylon (Tropilon-Plus™)membrane for 1 hr at 90V at 4° C. Blots were incubated in BlockingBuffer (BB-1 0.2% l-Block™, 0.1% Tween-20 in PBS! was used for PVDF andBB-2 3% l-Block™, 0.1% Tween-20 in PBS! was used for nylon) for 30minutes. Blots were then incubated with rabbit polyclonal antihumantransferrin (Boehringer/Mannheim cat #615 015; diluted 1:5000 in theappropriate Blocking Buffer) for 30 minutes, and then washed twice for 5minutes (PVDF with BB-1, and nylon with Wash Buffer 0.1% Tween-20 inPBS!). Next, blots were incubated with goat anti-rabbit IgG alkalinephosphatase conjugate (Tropix; diluted 1:10,000 in the appropriateBlocking Buffer) for 30 minutes, and then washed twice for 5 minutes asabove. Blots were then washed twice for 5 minutes in Assay Buffer (0.1MDEA, 1 mM MgCl₂, pH 10.0). Finally, blots were incubated with 0.25 mMCSPD or CDP-Star (diluted in Assay Buffer) for 5 minutes. Blots weredrained of excess substrate solution, placed in plastic report covers,and exposed to Kodak XAR-5 film. Results obtained on nylon and on PVDFmembranes are shown in FIGS. 4-5.

Chemiluminescent Detection of a single Copy Yeast Gene on Nylon Membrane

Total genomic DNA from the yeast Saccharomyces cerevisiae was digestedwith EcoR 1 and Bgl 11 restriction endonucleases. 5.0, 0.5, and 0.05 μgquantities of each DNA digest were separated by electrophoresis in ahorizontal 0.8% agarose 1× TBE gel. Following electrophoresis, the gelwas soaked in 1.5M NaCl, 0.5M NaOH for 45 minutes to denature the DNAand then incubated in neutralization buffer (1M Tris, 1.5M NaCl, pH 7.4)for 45 minutes. DNA was transferred to positively charged nylonmembranes (Tropilon-Plus™) by overnight capillary transfer using 20×SSC. The membranes were air dried and the DNA was UV cross linked to themembranes at 120 mJ/cm². A 1 kb Bgl 11 fragment, excised from the yeastgene RPB 1, was gel purified and biotinylated by a random primingreaction incorporating biotin-14-dCTP. The membranes were prehybridizedfor 30 minutes at 68° C. in hybridization solution (7% SDS, 0.25M Na₂PO₄, 1.0 mM EDTA), hybridized overnight at 68° C. with 5 ml of freshhybridization solution containing 5 ng/ml of denatured probe and thenremoved from the hybridization solution and washed as follows: twice for5 minutes with 2× SSC, 1% SDS at room temperature; twice for 15 minutesin 0.1×SSC, 1% SDS at 68° C.; and twice for 5 minutes in 1× SSC at roomtemperature. The membranes were then incubated for 10 minutes inblocking buffer (0.2% cassein, 0.5% SDS, PBS), and 20 minutes with1:5000 dilution AVIDx-AP™ in blocking buffer. They were then washed inblocking buffer for 5 minutes, three times for 10 minutes in wash buffer(0.5% SDS, PBS), and twice for 2 minutes in assay buffer (0.1Mdiethanolamine, 1.0 mM MgCl₂, pH 10.0) followed by incubation for 5minutes in 0.25 mM dioxetane in assay buffer. After draining excesssubstrate solution, the membranes were wrapped in plastic and exposed toX-ray film. 60 minute exposures taken 10 minutes, 70 minutes, and 19hours respectively after substrate incubation are reflected in thefollowing exposures. Comparisons of CSDP and CDP-Star are shown in FIGS.6, 7 and 8.

Chemiluminescent Detection of DNA Sequence Ladders

DNA sequencing reactions were performed with the Tropix SEQ-light™ kitusing biotinylated (-20) universal primer and single stranded M13 mp18template DNA. Reaction products were separated on a 6% polyacrylamide 8Murea gel, transferred to Tropilon-Plus™ nylon membranes by capillaryaction, and UV crosslinked to the membrane (total irradiation -120mJ/cm²). Chemiluminescent detection was performed by incubating themembrane for 10 minutes in blocking buffer (0.2% i-Block™, 0.5% SDS,PBS), for 20 minutes in conjugate solution (1/5000 dilution of Avidx-APstreptavidin alkaline phosphatase conjugate in blocking buffer), thenwashing 1×5 minutes with blocking buffer, 3×5 minutes with wash buffer(0.5% SDS, PBS), and 2×2 minutes with assay buffer (0.1M diethanolamine,1 mM MgCl₂, pH 10). Each membrane strip was incubated for 5 minutes witheither 0.25 mM CSPD or CDP-Star in assay buffer. All steps wereperformed at room temperature in a large heat sealed plastic bag withmoderate shaking (140-170 rpm). Comparison of CSPD and CDP-Star at threetime points is provided. The time after incubation with substrate andexposure time to Kodak XAR-5 x-ray film are indicated on FIG. 9.

Additional testing reflects values such as quantum yield (performed byan independent laboratory according to the procedure listed below),T_(1/2) and the emission wavelength maxima. These dioxetanes areidentified by number, and in the tables following after the number, theidentity of the substituent on the adamantyl ring, if any followed bythe identity of the Z substituent is given. In the compounds tested, Xis phosphate. Values for quantum yield and T_(1/2) are obtained both forthe dioxetane alone in 0.1 molar DEA, and in the presence of anenhancement agent, Sapphire II.

Protocol for Quantum Yields Determination

500 μL of 3.2×10⁻⁴ M solution of a dioxetane in 0.1M DEA, pH 10.0 wasplaced in a 12×75 mm tube, at 20° C. The solution was equilibrated to20° C. in a refrigerated water bath for 10 minutes. 2 μL of alkalinephosphatase suspension was added to the tube containing dioxetane andimmediately vortexed for 1 sec and placed in the 20° C. water bath. Thetube was then placed in MGM Optocomp® luminometer and the light signalwas measured at 1 sec integration times. After the light signal wasmeasured, the tube was placed back into the 20° C. water bath and themeasurement was repeated. The total counts for the dioxetane weredetermined from the intensity data. Total counts observed for a givenconcentration of dioxetane is the product of Photon Detection Efficiency(PDE) of the luminometer, the quantum yield of dioxetane and the numberof molecules capable of emitting light (concentration ofdephosphorylated dioxetanes). PDE for the MGM optocomp I luminometer wasdetermined to be 2.56×10⁻³, measured with a Biolink® absolute standardand utilizing the known spectral response of the luminometer's PMT andthe known emission spectrum of the dioxetanes. The quantum yield iscalculated by dividing the total counts measured by the PDE and theconcentration of the dioxetane.

Calculation of Half Life or Half Time to Steady State Light Emission

From the Turner luminometer readout, the maximum signal was measured.The maximum signal minus the Turner light unit readings at 30, 150, 300,or 600 second intervals was calculated and graphed vs. time in seconds.From the graphs, an exponential equation was calculated to determine thehalf life.

The half lives of the dioxetanes were also determined directly from theTurner luminometer printouts.

Emission Maxima

To 2 ml of a pH 10 solution of 0.4 mM dioxetane, 0.1M diethanolamine, 1mM MgCl₂ was added 9.9×10⁻¹¹ M alkaline phosphatase. The solution wasequilibrated 5 minutes in a Spex Fluorolog Fluorimeter and then scanned5 times at 0.5 sec/nm for chemiluminescent emission. Thechemiluminescence emission wavelength maximum was recorded.

Chemiluminescent DNA Sequencing

DNA sequencing with chemiluminescent detection was performed asdescribed in the Tropix SEQ-Light™ protocol. Briefly, DNA sequencingreactions were initiated with biotinylated primers using M13 singlestranded phage DNA as a template. The reactions were separated by 8Murea denaturing PAGE, transferred horizontally to Tropilon-Plus nylonmembrane by capillary action, and cross-linked to the membrane byexposure to UV light using a Spectronics SpectroLinker XL-1500 at 200mJ/cm². The membranes were incubated with blocking buffer (0.2%I-Block™, 0.5% sodium, dodecyl sulphate/SDS, in phosphate bufferedsaline/PBS (20 mM sodium phosphate, pH 7.2, 150 mM NaCl!) for 10minutes, incubated with a 1/5000 dilution of Avidx-APstreptavidin-alkaline phosphatase in blocking buffer for 20 minutes,washed for 5 minutes in blocking buffer, washed 3×5 minutes with washbuffer (0.5% SDS, PBS), washed 2×5 minutes with assay buffer (0.1Mdiethanolamine, 1 mM MgCl₂ pH 10), and then incubated with dioxetanesolution (either CSPD, 140-17 or 128-87 diluted to 0.25 mM in assaybuffer) for 5 minutes. The membranes were drained, sealed in a plasticfolder and exposed to Kodak XAR-5 X-ray film. For the dioxetane 128-87,the exposure time was 70 minutes and for 140-17, 80 minutes, both 65minutes after substrate addition. For the comparison of dioxetane 128-87versus CSPD, the membrane exposure time was 5 minutes after a 24 hourincubation with substrate. FIGS. 10 and 11. The details of this type ofprotocol are reflected in Tropix SEQ-Light™ DNA sequencing system,commercially available from Tropix, Inc.

0.1M DEA, pH 10, 25° C.

Dioxetane concentration 3.7×10⁻⁷ M to 6×10⁻⁶ M

    ______________________________________                                                     Quantum                                                          Compound     Yield       T 1/2 (min)                                                                             λL em                               ______________________________________                                        128-70 (H, 5-Cl)                                                                           1.4 × 10.sup.-4                                                                     35.55     471                                        128-87 (Cl, 5-Cl)                                                                          1.2 × 10.sup.-4                                                                     9.03      470                                        140-20 (H, 5-OMe)                                                                          1.5 × 10.sup.-5                                                                     1.55      476                                        140-17 (Cl, 5-OMe)                                                                         2.3 × 10.sup.-5                                                                     1.09      475                                        140-62 (H, 6-OMe)                                                                          1.1 × 10.sup.-6                                                                     2.4       490                                        140-73 (Cl, 6-OMe)                                                                         6.8 × 10.sup.-7                                                                     2.0       487                                        AMPPD        5.2 × 10.sup.-5                                                                     2.1       477                                        CSPD         5.2 × 10.sup.-5                                                                     1.6       475                                        ______________________________________                                    

0.09M DEA+0.1% Sapphire II, pH 9.95, 25° C.

Dioxetane concentration 1.8×10⁻⁷ M to 6.1×10⁻⁹ M

    ______________________________________                                                         Quantum                                                      Compound         Yield    T 1/2 (min)                                         ______________________________________                                        128-70 (H, 5-Cl) 5.2 × 10.sup.-2                                                                  172                                                 128-87 (Cl, 5-Cl)                                                                              3.5 × 10.sup.-2                                                                  70.6                                                140-20 (H, 5-OMe)                                                                              2.4 × 10.sup.-3                                                                  4.34                                                140-17 (Cl, 5-OMe)                                                                             1.9 × 10.sup.-3                                                                  1.1                                                 140-62 (H, 6-OMe)                                                                              3.8 × 10.sup.-5                                                                  6.49                                                140-73 (Cl, 6-OMe)                                                                             5.5 × 10.sup.-5                                                                  2.22                                                AMPPD            6.4 × 10.sup.-4                                                                  9.2                                                 CBPD               6 × 10.sup.-3                                                                  3.5                                                 ______________________________________                                    

To demonstrate positively the interaction of the dioxetane, or at leastthe excited-state emitter, with enhancement agents of the type known foruse in connection with dioxetanes, the wavelength for the emissionmaximum was detected in the absence of any enhancement agent, in thepresence of BDMQ, and on a nylon membrane. The data are set forth in thefollowing table.

    ______________________________________                                               Emission Max. nm                                                       Dioxetane                                                                              No Accmon     +BDMQ    On Nylon                                      ______________________________________                                        128-70   471           463      461                                           128-67   470           464      459                                           140-20   476           466      461                                           140-17   475           464      463                                           140-62   490           482      477                                           140-73   487           479      481                                           ______________________________________                                    

DOT BLOT ASSAYS

As noted above, the dioxetanes of this invention are suitable for use indot blot assays. The dioxetanes synthesized according to the synthesisroute described above were employed in dot blot assays. In confirmationof the absence of chemiluminescence of the dioxetanes bearing a Zsubstituent at the six position, it should be noted that Compound 140-62gave a consistent absence of signal, or, under optimum conditions, abarely detectable signal. Similarly, the dioxetane with the methoxysubstituent at the six position with a chlorine substituent on theadamantyl ring, 140-73, gave no signal in dot blot assay, againconfirming the lack of chemiluminescent activity in six-substitutedmetaphosphate phenyl dioxetanes. FIGS. 12-21.

Nitrocellulose and nylon membranes were spotted with a biotinylated 35base oligonucleotide probe. The probe was diluted in 1× SSC to yield astarting dilution of 210 pg. Successive 1:2 dilutions of the startingdilution were spotted on the membranes, 12 spots total. The membraneswere dried, subjected to optimum U.V. crosslinking (120 mJ/cm²), blockedfor 30 minutes in blocking buffer (nitrocellulose: 0.2% I-Block, 0.1%Tween-20, 1× PBS; nylon: 0.2% I-Block, 0.5% SDS, 1× PBS), incubated 20minutes in a 1/5000 dilution of streptavidin-alkaline phosphataseconjugate diluted in blocking buffer, and washed as follows: 1×5 minutesin blocking buffer; 3×5 minutes in 1× PBS, 0.3% Tween-20(nitrocellulose) or 3×5 minutes in 1× PBS, 0.5% SDS (nylon); 2×5 minutesin substrate buffer (0.1M diethanolamine, 0.1 mM MgCl₂, pH 10); 1×5minutes in a 1/20 dilution of Nitro-Block (Tropix, Inc. Bedford, Mass.)diluted in substrate buffer (Nitrocellulose Experiment Only); and 2×5minutes in substrate buffer (Nitrocellulose Experiment Only). Themembranes were incubated with 0.25 mM dioxetane diluted in substratebuffer for 5 minutes. Several membranes in both the nitrocellulose andnylon experiments were incubated with 0.25 mg/ml Calfax DB-45, Calfax10L-45 or Calsoft T-60 (Pilot Chemical Company, Los Angeles, Calif.),1.0 mg/ml Tween-20, 1.0 mg/ml Nitro-Block, and 0.25 mM dioxetane dilutedin substrate buffer for 5 minutes. These membranes were not subjected toa 1/20 dilution of Nitro-Block. The membranese were then exposed tox-ray film and developed.

Thus, as can be seen from the results above, electron withdrawing groupsadded to the aromatic ring of the dioxetane alter the kinetics of lightemissions while tending to increase the chemiluminescent signal. Incontrast, electron-donating groups accelerate T_(1/2) apparently byfacilitating electron transfer from the oxygen, through the aromaticgroup, to the dioxetane. Thus, by proper selection of the nature andability of the electron-donating or electron-withdrawing Z substituent,and simultaneous selection of the appropriate substituent for theadamantyl ring, if desired, dioxetanes of specific characteristics,including optimized signal intensity, optimized speed, specific emissionwavelength, and the like, can be obtained.

These dioxetanes can be used for assays of all types in which an enzymecapable of cleaving the dioxetane is present alone or can be attached toone element of the ultimate complex which the analyte, if present, willform. Conventional assay formats are known to those of skill in the art,and are described in the patents set forth above in the Background ofthe Invention. Exemplary disclosure of suitable assays appears in U.S.Pat. No. 5,112,960, and the same is incorporated herein by reference.The assay format, per se, save for the enhanced performance therein bythe dioxetanes of this invention, does not constitute an aspect of theinvention.

The dioxetanes of this invention, as well as the intermediatestherefore, have been disclosed by reference to both generic descriptionand specific embodiment. Additionally, dioxetane performance has beendescribed generally, and exemplified. The examples are not intended aslimiting, and should not be construed as such. Variations in substituentpattern, identity, and the like, consistent with the disclosure willoccur to those of ordinary skill in the art. Such variations andmodifications remain within the scope of the invention, save as excludedby the positive limitations set forth in the claims below.

What is claimed is:
 1. A dioxetane of the formula (I): ##STR6## whereinY¹ and Y² are each independently H, a hydroxyl group, a halogen, anunsubstituted lower alkyl group, a hydroxy lower alkyl group, a halolower alkyl group, a phenyl group, a halo phenyl group, an alkoxy phenylgroup, an alkoxy phenoxy group, a hydroxy alkoxy group, a cyano group,an amide group, an alkoxy group or a carboxyl group,wherein R is C1-20alkyl, aryl or aralkyl, wherein X is an enzyme-labile group selectedfrom the group consisting of a phosphate, galactoside, acetate,1-phospho-2,3-diacylglyceride, 1-thio-D-glucoside, adenosinetryphosphate, adenosine diphosphate, adenosine monophosphate, adenosine,α-D-glucoside, β-D-glucoside, β-D-glucuronide, α-D-mannoside,β-D-mannoside, β-D-fructofuranoside, β-glucosideuronate,P-toluenesulfonyl-L-arginine ester, and P-toluenesulfonyl-L-arginineamide, and wherein Z is an electron-active group selected from the groupconsisting of electron withdrawing groups and electron-donating groupsand occupies the four or five position on the phenyl ring.
 2. Thedioxetanes of claim 1, wherein Z is Cl, each of Y¹ and Y² is H, and R isan alkyl group substituted so as to enhance the solubility of thedioxetane in water.
 3. The dioxetane of claim 2, wherein Z is chlorineat the four and five position and wherein R is haloalkyl, dihaloalkyl ortrihaloalkyl.
 4. The dioxetane of claim 3, wherein Z is chlorine and Ris triflouroethyl.
 5. The dioxetane of claim 4, wherein Z is in the fourposition.